1. Field of the Invention
The present invention relates to homogeneous immunoassays for analytes and compositions of matter that are useful in conducting such immunoassays. Homogeneous immunoassays have the advantage of not requiring separation steps. Such assays, however, are limited by the difficulty of selecting antibodies which will modulate the activity of a label that is normally bound to the antibodies or an analog of the analyte.
The present invention relates to methods for immunoassay of analytes employing mutant glucose-6-phosphate dehydrogenase (G6PDH) enzymes as labels. In particular, the invention relates to the use of conjugates of an analyte and a mutant NAD+ dependent G6PDH of bacterial origin differing from any precursor G6PDH by the deletion, substitution, or insertion, or any combination thereof of at least one amino acid per subunit. The invention also involves the construction of several mutations in a precursor glucose-6-phosphate dehydrogenase (G6PDH) enzymes. Typically, the mutations involve deletion or substitution of one or more lysine residues, or introduction of one or more cysteine residues by insertion of cysteine to a precursor G6PDH or substitution of precursor G6PDH amino acid residues with cysteine. The present invention also relates to conjugates of the subject enzymes and specific binding pair members, cell lines producing the subject enzymes, DNA sequences encoding the subject enzymes, and plasmids containing DNA encoding the subject enzymes and designed to allow a host cell to produce the subject enzymes.
2. Brief Description of the Related Art
Adams, M. J., H. R. Levy, and K. Moffat; 1983; Crystallization and preliminary X-ray data for glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides; J. Bio. Chem. 258:5867-5868; discloses the crystallization and preliminary X-ray data for glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. 
Barnell, W. O., K. C. Yi, and T. Conway; 1990; Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism; J. Bacteriology 172:7227-7240; discloses cloning, sequence and organization of Zymomonas mobilis genes encoding glycolytic pathway enzymes, including glucose-6-phosphate dehydrogenase. The information is said to be useful as a tool for studying the contribution of gene expression to flux control at each step of the pathway.
Bhadbhade, M. M., M. J. Adams, T. G. Flynn, and H. R. Levy; 1987; Sequence identity between a lysine-containing peptide from Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and an active site peptide from human erythrocyte glucose-6-phosphate dehydrogenase; FEBS Lett. 211:243-246; discloses the sequence identity between a lysine-containing peptide from Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and an active site peptide from human erythrocyte glucose-6-phosphate dehydrogenase.
Gasser, F., and M. Hontebeyrie; 1977; Immunological relationships of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides NCDO 768 (=ATCC 12291); Int. J. Systematic Bact. 27:6-8; discloses the immunological cross-reactivity patterns of antibodies capable of recognizing Leuconostoc mesenteroides with various Leuconostoc strains
Haghighi, B., T. G. Flynn, and H. R. Levy; 1982; Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides; Isolation and sequence of a peptide containing an essential lysine; Biochemistry 21:6415-6420; discloses the interaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with pyridoxal 5xe2x80x2-phosphate and sodium borohydride.
Heilmann, H. J., H. J. Migert, and H. G. Gassen; 1988; Identification and isolation of glucose-6-phosphate dehydrogenase genes of Bacillus megaterium M1286 and their expression in Escherichia coli; Eur. J. Biochem. 174:485-490; discloses the identification and isolation of glucose dehydrogenase genes of Bacillus megateriumM1286 and their expression in Escherichia coli. 
Hey, Y., and P. D. G. Dean; 1983; Tandem dye-ligand chromatography and biospecific elution applied to the purification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides; Biochem. J. 209:363-371; discloses the purification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. 
Hontebeyrie, M.; and F. Gasser; 1975; Comparative immunological relationships of two distinct sets of isofunctional dehydrogenases in the genus Leuconostoc; Int. J. Systematic Bact. 25:1-6; discloses the immunological cross-reactivity patterns of antibodies capable of recognizing Leuconostoc lactis with various Leuconostoc strains and heterofermentative lactobacilli.
Ishaque, A., M. Milhausen, and H. R. Levy; 1974; On the absence of cysteine in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides; Biochem. Biophys. Res. Commn. 59:894-901; discloses the complete lack of cysteine in Leuconostoc mesenteroides. 
Jarsch, M., and G. Lang; Cloning and overexpression of glucose-6-phosphate dehydrogenase from Leuconostoc dextranicus; Canadian Patent Application Number 2,045,838 A1 (published Jan. 31, 1992); discloses recombinant glucose-6-phosphate dehydrogenase enzymes derived from Leuconostoc dextranicus having improved temperature stability.
Jeffery, J., L. Hobbs, and H. Jxc3x6rnvall; 1985; Glucose-6-phosphate dehydrogenase from Saccharomyces cerevisiae: characterization of a reactive lysine residue labeled with acetylsalicylic acid; Biochem. 24:666-671: discloses the characterization of a reactive lysine residue that reacts with acetylsalicylic acid.
Jeffery, J., I. Wood, A. Macleod, R. Jeffery, and H. Jxc3x6rnvall; 1989; Glucose-6-phosphate dehydrogenase; Biochem. Biophys. Res. Commn. 160:1290-1295; discloses the characterization of a reactive lysine residue in the Pichia jadirnii glucose-6-phosphate dehydrogenase enzyme. The information is said to reveal a limited structural variation in a functionally significant segment of the enzyme.
Lee, W. T., T. G. Flynn, C. Lyons, and H. R. Levy; 1991; Cloning of the gene and amino acid sequence for glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides; J. Bio. Chem. 266:13028-13034; discloses the cloning of the Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase gene and the amino acid sequence of the enzyme, derived from partial sequencing of the DNA. The information is said to be useful for site-directed mutagenesis studies of those structural features of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase that allow for NAD+ binding and utilization.
Lee, W. T., and H. R. Levy; 1992; Lysine-21 of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase participates in substrate binding through charge-charge interaction; Protein Science 1:329-353; discloses the purification and kinetic characterization of Lys-21-Arg and Lys-21-Gln mutants of glucose-6-phosphate dehydrogenase in order to determine the function of Lys-21.
Levy, H. R.; 1979; glucose-6-phosphate dehydrogenases; Advances in Enzymology 48:97-192; discloses the isolation, structure, and catalytic activity of glucose-6-phosphate dehydrogenases.
Levy, H. R., and W. T. Lee; Cloned Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase genes and methods of making same; International Patent Application Number PCT/US91/07715 (International publication WO 92/07078, Apr. 30, 1992) discloses the isolation, PCR amplification and cloning of a gene for glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides into plasmid pUC19 for expression in E. coli. 
Murphy, N. B., D. J. McConnell, and T. F. R. Schwarz; 1987; Expression of the gene for NAD+-dependent glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides cloned in Escherichia coli K-12; J. Bacteriology 169:334-339; discloses the expression of the gene for NAD+-dependent glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides cloned in Escherichia coli K-12.
Olive, C., and H. R. Levy; 1967; The preparation and some properties of crystalline glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides; Biochemistry 6:730-736; discloses the purification of glucose-6-phosphate dehydrogenase from extracts of Leuconostoc mesenteroides. 
Rowley, D. L., and R. E. Wolf; 1991; Molecular characterization of the Escherichia coli K-12 zwf gene encoding glucose-6-phosphate dehydrogenase; J. Bacteriology 173:968-977; discloses cloning and sequencing of Escherichia coli K-12 glucose-6-phosphate dehydrogenase. The information is said to be useful in the search for an internal complementary sequence that functions as a cis-acting antisense RNA by forming a long-range secondary structure that sequesters the ribosome binding site.
Rubenstein, K. E., and R. K. Leute; Antibody Steric Hindrance Immunoassay with Two Antibodies; U.S. Pat. No. 3,935,074, Jan. 27, 1976 (filed Dec. 17, 1973); discloses an antibody steric hindrance immunoassay using two antibodies.
Rubenstein, K. E., and E. F. Ullman; Enzyme amplification Assay; U.S. Pat. No. 3,817,837, Jun. 18, 1974 (filed Nov. 6, 1972); discloses homogeneous immunoassays.
Skold, C., I. Gibbons, D. Gould, and E. F. Ullman; 1987; Monoclonal antibodies to glucose-6-phosphate dehydrogenase (G6PDH) form cyclic 1:1 complexes with G6PDH and act as regulatory subunits; J. Immunology 138:3408-3414; and Skold, C., D. R. Gould, and E. F. Ullman; Methods for modulating ligand-receptor interactions and their application; U.S. Pat. No. 4,727,022, Feb. 23, 1988 (filed Mar. 14, 1984); discloses the preparation of inhibitory antibodies to G6PDH from L. mesenteroides. 
Yoshida, R. A., and E. T. Maggio; Antienzyme Homogeneous Competitive Binding Assay; U.S. Pat. No. 4,233,401, Nov. 11, 1980 (filed Jul. 14, 1977); discloses a protein binding assay for a member of an immunological pair whereby an enzyme-ligand conjugate is employed in combination with an enzyme inhibitor that can be an antibody.
One aspect of the present invention relates to methods for homogeneous immunoassay of an analyte in a sample suspected of containing the analyte. The methods comprise the steps of: (1) combining in a liquid medium: (a) the sample to be assayed, (b) a conjugate of (i) an analyte analog and (ii) a mutant NAD+ dependent G6PDH differing from any precursor G6PDH by the deletion, substitution, or insertion or any combination thereof of at least one amino acid per subunit, (c) a receptor for the analyte, and (d) substrates for the G6PDH; (2) determining the enzymatic activity of the G6PDH in the medium; and (3) comparing the enzymatic activity to the enzymatic activity observed with a sample containing the analyte.
Another aspect of the present invention relates to methods for determining the amount of a specific binding pair (sbp) member in a sample suspected of containing the sbp member. The methods comprise the steps of: (a) combining in an assay medium: (1) the sample, (2) a conjugate of an enzyme and an analog of the sbp member, wherein the enzyme is a mutant bacterial glucose-6-phosphate dehydrogenase (G6PDH) having at least two amino acid mutations per subunit as compared to precursor G6PDH, and (3) an sbp partner of the sbp member capable of binding the conjugate, and (b) determining the activity of the enzyme.
Another aspect of the present invention relates to methods for determining the presence or amount of an analyte in a sample suspected of containing the analyte. The methods comprise the steps of: a) combining in an assay medium: 1) the sample, 2) a conjugate of an analyte analog and an enzyme, wherein the enzyme is a mutant glucose-6-phosphate dehydrogenase (G6PDH) derived from an organism selected from the group consisting of Leuconostoc mesenteroides, Leuconostoc citreum, Leuconostoc lactis, and Leuconostoc dextranicus wherein the G6PDH has at least one amino acid mutation per subunit as compared to precursor G6PDH wherein at least one of the mutations comprises the introduction of a cysteine residue proximate to an epitope recognized by an inhibitory anti-G6PDH antibody capable of simultaneously binding to two of the subunits within the same G6PDH molecule, 3) an antibody capable of binding the analyte and the analyte analog conjugate, and 4) substrates for the enzyme; and b) measuring the activity of the enzyme.
Another aspect of the present invention relates to improved methods for determining the presence of a ligand in a sample suspected of containing the ligand. The assay to be improved includes the steps of: a) bringing together in an aqueous medium: 1) the sample, 2) enzyme-bound-ligand, and 3) receptor capable of binding to the ligand and the enzyme-bound-ligand, wherein the receptor is at a concentration sufficient to substantially change the enzymatic activity of the enzyme-bound-ligand in the absence of the ligand; b) determining the enzymatic activity of the enzyme-bound-ligand in the medium; and the improvement comprises employing as the enzyme a mutant glucose-6-phosphate dehydrogenase (G6PDH) having at least two amino acid mutations as compared to a precursor G6PDH.
Another aspect of the present invention relates to compositions comprising a specific binding pair member conjugated to a mutant NAD+ dependent bacterial glucose-6-phosphate dehydrogenase (G6PDH) having at least one amino acid mutation per subunit as compared to precursor G6PDH.
Another aspect of the present invention relates to a mutant glucose-6-phosphate dehydrogenase (G6PDH) enzyme that is the expression product of a mutated DNA sequence encoding a glucose-6-phosphate dehydrogenase, the mutant DNA sequence being derived from a precursor glucose-6-phosphate dehydrogenase by the deletion, insertion or substitution of one or more amino acids in the precursor glucose-6-phosphate dehydrogenase. Preferably, the G6PDH is an NAD+ dependent bacterial G6PDH, more preferably the mutant DNA sequence is derived from a precursor glucose-6-phosphate dehydrogenase by the deletion, insertion or substitution of two or more amino acids in the precursor glucose-6-phosphate dehydrogenase.
Another aspect of the present invention relates to mutant glucose-6-phosphate dehydrogenase (G6PDH) enzymes having at least one mutation per subunit as compared to precursor G6PDH wherein the mutation is proximate to an epitopic site recognized by an anti-G6PDH antibody capable of inhibiting the activity of the precursor G6PDH.
Another aspect of the present invention relates to mutant DNA sequences encoding such glucose-6-phosphate dehydrogenase (G6PDH) enzymes. These mutant DNA sequences are derived from a precursor DNA sequence which encodes a naturally-occurring or recombinant precursor enzyme. The mutant DNA sequences are derived by modifying the precursor DNA sequence to encode the substitution, deletion or insertion of at least one amino acid residue encoded by the precursor DNA sequence. These recombinant DNA sequences encode glucose-6-phosphate dehydrogenase mutant enzymes having a novel amino acid sequence.
Further the invention relates to expression vectors containing such mutant glucose-6-phosphate dehydrogenase DNA sequences as well as host cells transformed with such vectors which are capable or producing such mutant enzymes.
Another aspect of the present invention relates to improved assay reagents for use in the determination of an analyte in a sample suspected of containing the analyte. The assay reagents include an analyte-label conjugate. The improvement comprises employing as the label mutant NAD+ dependent glucose-6-phosphate dehydrogenase (G6PDH) enzymes having at least one mutation per subunit as compared to precursor G6PDH wherein the mutations are proximate to an epitopic site recognized by an anti-G6PDH antibody capable of inhibiting the activity of the precursor G6PDH.
Another aspect of the present invention relates to kits for conducting an assay for the determination of a specific binding pair (sbp) member. The kits comprise in packaged combination an sbp partner of the sbp member and a composition which comprises the sbp member or an analog of the sbp member conjugated to a mutant NAD+ dependent bacterial glucose-6-phosphate dehydrogenase (G6PDH) having at least one amino acid mutation per subunit as compared to precursor G6PDH.